Formation of immobilized biological layers for sensing

ABSTRACT

The invention is directed to enzyme immobilization compositions comprising: one or more enzymes, a humectant, an acrylic-based monomer, a water-soluble organic photo-initiator and a water-soluble acrylic-based cross-linker in a substantially homogeneous aqueous mixture. The invention is also directed to methods for forming sensors comprising such compositions and to apparati for forming arrays of immobilized layers on an array of sensors by dispensing such compositions onto a substrate.

This application is a divisional of U.S. application Ser. No. 11/961,550filed on Dec. 20, 2007, the entire contents and disclosure of which ishereby incorporated by reference.

FIELD OF THE INVENTION

An apparatus and method for manufacture of immobilized biological layersare disclosed. The technology is capable of being used for sensinganalytes in liquid samples in the point-of-care clinical diagnosticfield and beyond. A curable composition of matter for the formation ofimmobilized biological layers is also disclosed.

BACKGROUND OF THE INVENTION

The development of miniaturized sensors for the measurement ofbiologically significant analyte species in biological fluids isbecoming increasingly important, particularly because of the need forincreasingly smaller devices that permit the measurement of such analytespecies in the field or in the home. Notwithstanding advances in thefield of sensor fabrication, there still exist major challenges in theminiaturization and fabrication of such sensors. One such challenge isthe degree of complexity involved with the mass production ofcommercially viable sensors that comprise biological active molecules.Of major concern is the compatibility of the inherently harsh physicaland chemical processes associated with existing semiconductormanufacturing methods, with sensitive organic compounds and labilebiologically active molecules, both of which comprise parts of afunctioning biological sensor. Another major challenge surrounding theminiaturization and fabrication of such sensors is the production ofsensors that are sensitive and that can be made in mass quantities witha high degree of reproducibility. There is therefore a need forprocesses for forming sensors that take into account the sensitivity ofthe biologically active molecules used in the sensors, as well as theneed for a highly uniform sensor when the sensor is produced in largequantities.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood in view of the followingnon-limiting figures, wherein:

FIG. 1 shows a topological elevation cross-sectional view of a bloodurea nitrogen (BUN) sensor fabricated on a silicon wafer in combinationwith a reference electrode;

FIGS. 2( a)-(c) show plan views of a BUN sensor on a silicon chip (2mm×3 mm) at different steps of manufacture, as follows: FIG. 2( a) thebare silver-silver chloride electrode 353; FIG. 2( b) as for (a) with amicrodispensed ammonium ion-selective membrane 355; FIG. 2( c) as for(b) with an ultraviolet spot-cured acrylamide urease enzyme layer 356.

FIGS. 3( a)-(c) show electron micrograph views of finished BUN sensors,as follows: FIG. 3( a) shows an immobilized enzyme layer formed by theconventional ELVACE process (a vinyl acetate ethylene copolymer composedof hydrophilic and hydrophobic domains) process; FIG. 3( b) a UVspot-cured acrylamide urease enzyme layer with the desired uniform domedshape; and FIG. 3( c) a UV flood cured acrylamide urease enzyme layer;

FIG. 4 shows a sensor output data (chronopotentiometric graph) for anacrylamide BUN sensor using the composition described in FIG. 14( a), ingoing from calibrant fluid to blood;

FIG. 5 shows a sensor correlation data for acrylamide BUN sensors (EIL)of the type shown in FIG. 4, in whole blood (WB) for both a heated andun-heated chip compared to the ELVACE based (wood glue) enzymeimmobilization membrane;

FIG. 6 shows a view of dispensing apparatus and UV spot-curingsubsystem;

FIG. 7 shows details of the spot-curing subsystem and the UV light box;

FIGS. 8( a)-(b) shows details of (a) the dispensing and (b) thespot-curing subsystems;

FIG. 9 shows process algorithm steps and timing for dispensing andspot-curing;

FIG. 10 is an isometric top view of a sensor cartridge cover;

FIG. 11 is an isometric bottom view of a sensor cartridge cover;

FIG. 12 is a top view of the layout of a tape gasket for a sensorcartridge;

FIG. 13 is an isometric top view of a sensor cartridge base;

FIG. 14( a)-(b) show a table of reagents for matrix with preferredactual mixture compositions where FIG. 14( a) shows the components forthe urease containing enzyme immobilization layer with urease as theonly enzyme, and FIG. 14( b) is similar to 14(a) with the addition ofcarbonic anhydrase;

FIG. 15( a)-(b) shows chronopotentiometric data of signals generatedfrom enzyme immobilization layers generated using (a) acrylamide,methyacrylamide, poly(ethylene glycol) acrylate (PEGA), andN-[3-(dimethylamino)propyl]-methacrylamide (DMAPMA) as monomers and1,4-bis(acryloyl)piperazine as dimer, (b) acrylamide as monomer and1,4-bis(acryloyl)piperazine, polyethylene glycol diacrylatepoly(ethylene glycol)diacrylate (PEGDA),N,N′-(1,2-dihydroxyethylene)bis-acrylamide (DHEBA) andtrimethylolpropane ethoxylate triacrylate (TMPETA) as dimers;

FIG. 16 shows chronopotentiometric data of signals generated from anacrylamide/1,4-bis(acryloyl)piperazine based enzyme immobilization layerafter different intensity and times of exposure to UV light at 310 nm,demonstrating no significant impact of a range of time and intensity ofexposures used in this experiment to UV light;

FIG. 17 shows data on the impact of the humectant at differentconcentrations with different shelf-life and storage conditions; and

FIG. 18 demonstrates the relatively minor effect of print thickness ofthe EIL membrane on sensor performance using five different aqueouscontrol fluids.

SUMMARY OF THE INVENTION

The present invention provides an apparatus and method for themanufacture of immobilized biological layers for use in sensors for themeasurement of biologically significant analyte species in biologicalfluids. The method and apparatus help produce devices with more uniformperformance characteristics across a large manufacturing lot. Anadditional valuable feature of the method and apparatus is that theyprovide biological layers that substantially resist swelling whencontacted with a liquid, such as, e.g., calibrant fluid, control fluidand blood. While not bound by any particular theory, it is believed thatthe biological layers produced using the method and apparatus resistswelling because there is a significant level of crosslinking in thelayers. It has been found that a membrane that resists swelling in thisway is desirable for the operation of the sensor, as biological layersthat exhibit significant swelling can give inconsistent signals and evendelaminate from the surface.

In one aspect, the invention is directed to a method of forming a sensorcomprising the steps of: (a) forming a substantially homogeneous aqueousmixture comprising one or more enzymes, an acrylic-based monomer, awater soluble organic photo-initiator and a water soluble acrylic-basedcross-linker in an aqueous mixture, (b) applying a controlled volume ofsaid mixture onto a base sensor sufficient to cover said base sensor,and (c) exposing said applied volume to sufficient UV radiation to forman immobilized enzyme layer adhered to said base sensor.

Optionally, the enzyme is selected from the group consisting of urease,glucose oxidase, lactate oxidase, creatinase, creatininase, sarcosineoxidase, catalase, carbonic anhydrase, NAD(P)H oxidase, cholesteroloxidase, alcohol oxidase, choline oxidase, glycerol-3-phosphate oxidase,thiamine oxidase, pyruvate oxidase, pyridoxal oxidase, D-amino acidoxidase, L-amino acid oxidase, alkaline phosphatase, horseradishperoxidase and combinations thereof. The monomer may, for example, beselected from acrylamide, methacrylamide,N-[3-(dimethylamino)propyl]methacrylamide, hydroxyethylmethacrylate andcombinations thereof. The organic photo-initiator may be selected from2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone, (insertmeans for claim 8) and combinations thereof. The cross-linker optionallyis selected from 1,4-bisacryloyl piperazine,N,N′-(1,2-dihydroxyethylene)bis-acrylamide,N,N′-bis(acryloyl)cystamine,N,N′-methylenebisacrylamide, ethylene glycol diacrylate,(+)-N,N′-diallyltartramide and combinations thereof. The base sensoroptionally is selected from the group consisting of electrode,ion-selective electrode, potentiometer electrode, amperometricelectrode, conductimetric electrode, enzyme electrode, biosensor,optical sensor, fiber optic sensor, surface acoustic wave sensor,evanescent sensor, surface plasmon resonance sensor and optical waveguide sensor. In a preferred embodiment, the enzyme is urease and thebase sensor is an ammonium ion-selective membrane.

The aqueous mixture optionally further comprises one or more stabilizingcomponents selected from the group consisting of pH buffer, disulfidebond reducing agent, divalent ion chelating agent, protease inhibitor,bovine serum albumin, salts, biocide and humectant. The aqueous mixtureoptionally comprises urease,2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone,bisacryloyl piperazine, glycerol and acrylamide monomer.

The method of applying the mixture may be by microdispensing acontrolled volume in the range of about 5 nL to about 1 μL. The methodof applying the mixture may be by means selected from the groupconsisting of spin-coating, dip-coating, spray coating, screen printing,ink-jet printing, laser printing, painting and contact printing.

The UV radiation may be, for example, in the wavelength range of about185 to 400 nm and optionally has an intensity in the range of about 100to 400 mW/cm². said UV radiation step is a spot cure performedimmediately after the dispensing step in a dispensing cycle. The UVradiation step optionally is a spot cure performed with a pre-selectedtime delay after the dispensing step, and may be performed for aduration of about 0.1 to 10 seconds.

In another embodiment, the invention is to a method of forming a ureasensor comprising the steps of: (a) forming a substantially homogeneousaqueous mixture comprising urease, glycerol,2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone,bisacryloyl piperazine and acrylamide monomer, (b) dispensing acontrolled volume of said mixture onto an ammonium ion-selectivemembrane sufficient to cover said membrane, and (c) exposing saiddispensed volume to sufficient UV radiation to form an immobilizedurease layer adhered to said membrane.

In another embodiment, the invention is to an apparatus for forming animmobilized enzyme layer on a substantially planar surface, theapparatus comprising a dispensing head for dispensing a controlledvolume, e.g., in the range of about 5 nL to about 1 μL, of aphotoformable enzyme-containing matrix at a pre-selected location onsaid surface and a UV radiation source, e.g., a mercury lamp, with aregistration and alignment means capable of focusing a beam of radiationonto an area substantially covering said pre-selected location at apredetermined time and for a predetermined duration, at a predeterminedintensity, after said matrix has been dispensed.

Optionally, the apparatus further comprises a step and repeat means forchanging the relative position of said surface with respect to saiddispensing head and said UV radiation source for forming an array ofimmobilized enzyme layers at a set of pre-selected locations.

The substantially planar surface optionally is selected from a siliconwafer, alumina wafer, liquid crystal substrate, glass substrate andplastic substrate and flexible plastic substrate. The dispensing headmay comprise, for example, a syringe needle with a reservoir for saidmatrix, and a displacement means for controlling the dispensed volumefrom said syringe onto said surface.

The photoformable matrix optionally is the composition comprising one ormore enzymes, a humectant, an acrylic-based monomer, a water solubleorganic photo-initiator and a water soluble acrylic-based cross-linkerin a substantially homogeneous aqueous mixture.

The pre-selected location preferably has an area in the range of about10 square microns to about 75 square millimeters. Optionally, thepre-selected location is substantially circular and has radialdimensions in the range of about 5 μm to about 5 mm.

Optionally, registration and alignment means permit the beam to befocused on a selected area of said surface and illuminate an area in therange of about 10 square microns to about 75 square millimeters. Acomputer program may control the timing and location of dispensingand/or the timing and location of the application of UV radiation withrespect to the timing of dispensing.

Optionally, the dispensing head is capable of dispensing a sequence ofcontrolled volumes of a photoformable matrix at a pre-selected set oflocations on said surface, and said UV radiation source is capable offocusing a beam of radiation onto an area substantially covering eachsaid pre-selected locations, in sequence at a predetermined time aftereach controlled volume is dispensed, for a predetermined duration.

In another embodiment, the invention is to an apparatus for forming anarray of immobilized enzyme layers on an array of sensors on asubstantially planar surface comprising: a dispensing head fordispensing a sequence of controlled volumes of a photoformableenzyme-containing matrix at a pre-selected set of locations on saidsurface, and a UV radiation source with a registration and alignmentmeans capable of focusing a beam of radiation onto an area substantiallycovering each said pre-selected location, in sequence at a predeterminedtime after each controlled volume is dispensed, for a predeterminedduration.

In another embodiment, the invention is to a method of forming animmobilized layer on a sensor on a substantially planar surfacecomprising the steps of: (a) dispensing a controlled volume of aphotoformable matrix at a pre-selected location on said surface, whereinthe photoformable matrix comprises a biologically active material; and(b) applying a UV radiation beam onto an area substantially coveringsaid pre-selected location, starting at a predetermined time after saidvolume has been dispensed and for a predetermined duration at apredetermined intensity, to form said immobilized layer.

The photoformable matrix optionally comprises one or more enzymes, anacrylic-based monomer, a water soluble organic photo-initiator and awater soluble acrylic-based cross-linker in an aqueous mixture. Thebiologically active material optionally is selected from the groupconsisting of protein, enzyme, antibody, antibody fragment, RNA, singlestranded DNA and double stranded DNA. The immobilized layer preferablyis an enzyme layer.

In another embodiment, the invention is to a method of forming an arrayof immobilized layers on an array of sensors on a substantially planarsurface comprising the steps of: (a) dispensing a sequence of controlledvolumes of a photoformable matrix at a pre-selected set of locations onsaid surface, and (b) applying a UV radiation beam onto an areasubstantially covering each said pre-selected location, in sequence,starting at a predetermined time, e.g., in the range of about 0.1 toabout 10 seconds, after each controlled volume has been dispensed, andapplying said radiation at a predetermined intensity for a predeterminedduration, e.g., in the range of about 0.1 to about 10 seconds, to formsaid immobilized array of layer. The UV radiation preferably is in thewavelength range of about 185 to 400 nm and the UV radiation intensitymay be in the range of about 100 to 400 mW/cm². The planar surfaceoptionally is a silicon wafer and the pre-selected set of locations isan array of sensors on said wafer. The UV radiation beam optionally isapplied to the Nth minus X pre-selected location while dispensing occursat the Nth pre-selected location, where X is equal to an integer from 1to 10.

In another embodiment, the invention is to a sensor, comprising anelectrode with a first layer covering said electrode comprising anion-selective membrane, and a second layer covering said ion-selectivemembrane comprising a UV cured matrix formed from a substantiallyhomogeneous aqueous mixture of one or more enzymes, a humectant, anacrylic-based monomer, a water-soluble organic photo-initiator and awater-soluble acrylic-based cross-linker.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the invention relates to an enzyme immobilizationcomposition comprising one or more enzymes, a humectant, anacrylic-based monomer, a water-soluble organic photo-initiator and awater-soluble acrylic-based cross-linker in a substantially homogeneousaqueous mixture.

Enzymes

The one or more enzymes included in the composition of the presentinvention may vary widely. In some embodiments, the one or more enzymescomprises urease. The enzyme urease is particularly well suited forincorporation into biosensors that quantify the blood urea nitrogen(BUN) content in an assay. BUN assays are useful in measuring the levelsof urea nitrogen, a waste product of protein metabolism that is clearedby the kidneys, in the blood. BUN assays therefore assess renalfunction. Clinically useful BUN values are 2-140 mg/100 mL (dL). Thecondition known as azotemia, i.e., increased BUN levels, can indicateimpaired renal function, congestive heart failure, dehydration, shock,hemorrhage into the gastrointestinal tract, stress, acute myocardialinfarction or excessive protein intake. Alternatively, decreased BUNvalues may indicate liver failure, malnutrition, anabolic steroid use,pregnancy and siliac disease.

Urea in whole blood is detected in a two-step process. First, the ureais enzymatically converted to the products NH₄ ⁺ and HCO₃ ⁻ in “theurease reaction” via a mechanism that is not well understood. The secondstep in the detection of urea is the potentiometric determination ofammonium ion activity by the NH₄ ⁺ ion-selective electrode (ISE). See,D. Freifelder, Physical Biochemistry: Applications to Biochemistry andMolecular Biology Chapter 4 (2d ed. 1982). The BUN sensor response,i.e., change in potential due to changes in the concentration of NH₄ ⁺,is calibrated at known levels of urea in blood. A plot of a sensorresponse curve, (chronopotentiometric graph in millivolts as a functionof time), thus can be used to indicate the concentration of ammonium ionwithin the sensor membrane, which provides an estimate indirectly of theurea concentration in the blood.

Since the enzymatic breakdown of urea by urease produces the species H⁺and CO₂ as byproducts from the decomposition of HCO₃ ⁻, the BUN contentcan be determined using sensors that detect changes in the H⁺ or CO₂.Detection of ammonium ion is preferred because of the relatively lowbackground concentration of ammonium ions in the blood. In contrast,blood has a significant background of H⁺, CO₂ and HCO₃. The productionof ions during the urease reaction also increases the conductivity ofthe sample, which can be detected with a conductivity sensor.

An ideal property of urease is that it has a low residual level ofassociated product (ammonium ions <0.00001 μmol/enzyme unit) and othernitrogenous compounds. The urease that is used in the enzymeimmobilization compositions of the present invention should ideally befree of contaminating proteases and should have specific activitiesgreater than 500 U/mg protein at 25° C. In addition, the enzyme shouldalso be of high purity. In some embodiments, the urease should have aK_(m) in the range of from about 1 to about 100 mM, e.g., from about 1to about 50 mM or from about 25 to about 75 mM, and preferably about 50mM. In addition, the urease should have a V_(max) greater than 16,000(micromol/ml/min). Finally, the urease should have a K_(cat) of about5×10⁵ min⁻¹ or greater.

In a preferred embodiment, the urease is Jack Bean urease (E.C. 3.5.1.5)(Biozyme Laboratories, San Diego, Calif.). Other sources of Jack BeanUrease (E.C. 3.5.1.5) include; (i) Sigma-Aldrich Canada Ltd. (Oakville,Ontario, Canada); (ii) Toyobo (Tokyo, Japan); (iii) WorthingtonBiochemical Corporation (Lakewood, N.J.), (iv) Genzyme Diagnostics(Cambridge, Mass.).

In some embodiments the enzyme immobilization compositions of thepresent invention comprise carbonic anhydrase and urease. Carbonicanhydrase converts bicarbonate formed by the urease reaction to carbondioxide, thereby increasing the rate of ammonium ion production asdescribed in U.S. patent application Ser. No. 11/216,041, the entiretyof which is incorporated herein by reference. The carbonic anhydrasethat is used in these compositions should ideally have low residuallevels of nitrogenous compounds, be free of contaminating proteases andshould otherwise be of high purity. In addition, the carbonic anhydraseshould have a specific activity greater than 2500 Wilbur-Andersonunits/mg protein at 0° C. (Wilber, K. M. and N. G. Anderson, Journal ofBiological Chemistry 176: 147-154 (1948)). In some embodiments, thecarbonic anhydrase should have a K_(m) value between 1 to 50 mM, wherethe preferred K_(m) is 1 to 5. In addition, the carbonic anhydraseshould have a V_(max) greater than 50 (microl/ml/min), preferably above10,000 (microl/ml/min). Finally, the carbonic anhydrase should have aK_(cat) value greater than 75 min⁻¹, preferably greater than 5×10⁵min⁻¹.

In a preferred embodiment, the carbonic anhydrase is bovine carbonicanhydrase (E.C. 4.2.1.1) (Sigma-Aldrich Canada Ltd., Oakville, Ontario,Canada; K_(m): 1.31 mM; V_(max): 64.4 micromol/ml/min; K_(cat): 76.24min⁻¹). Another source of Bovine carbonic anhydrase (E.C. 4.2.1.1) isWorthington Biochemical Corporation (Lakewood, N.J.).

Although in some preferred embodiments, the enzyme used in the enzymeimmobilization compositions of the present invention is urease, anyother enzyme that is compatible with the immobilization compositions canbe used individually or in combination with another enzyme (e.g., ureaseand carbonic anhydrase). In some embodiments, the enzyme can be selectedfrom the group consisting of glucose oxidase, lactate oxidase,creatinase, creatininase, sarcosine oxidase, catalase. NAD(P)H oxidase,cholesterol oxidase, alcohol oxidase, choline oxidase,glycerol-3-phosphate oxidase, thiamine oxidase, pyruvate oxidase,pyridoxal oxidase, D-amino acid oxidase, L-amino acid oxidase, urease,alkaline phosphatase, horseradish peroxidase and combinations thereof.It can be appreciated that the enzyme used determines the analyte thatis being sensed. Thus, for example, glucose oxidase can be used in asensor to detect glucose; lactate oxidase can be used to detect lactate;and a combination of urease and carbonic anhydrase can be used for thesimultaneous detection of BUN content.

Monomers, Photo-Initiators and Cross-Linkers

As discussed above, the enzyme immobilization compositions of thepresent invention comprise an acrylic-based monomer, a water-solubleorganic photo-initiator and a water-soluble acrylic-based cross-linker.In some embodiments, the acrylic-based monomer comprises an acrylamide.In other embodiments, the monomer comprises a methacrylamide,poly(ethylene glycol)acrylate,N-[3-(dimethylamino)propyl]methacrylamide, hydroxyethylmethacrylate, ormixtures thereof.

The organic photo-initiator can be any photo-initiator that is capableof polymerizing a monomer. In some embodiments, the photo-initiator isselected from the group consisting of2,6-bis(4-azidobenzylidene)cyclohexanone;2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone;4,4-diazidostilbene-2,2′-disulfonic acid disodium salt; ammoniumdichromate; 1-hydroxy-cyclohexyl-pentyl-keton (Irgacure 907);2-methyl-1[4-(methylthio)phenyl]-2-morpholinopropane-1-one (Irgacure184C); 2-hydroxy-2-methyl-1-phenyl-propane-1-one (Darocur 1173); a mixedphoto-initiator (Irgacure 500) of 50 wt % of Irgacure 184C and 50 wt %of benzophenone; a mixed initiator (Irgacure 1000) of 20 wt % ofIrgacure 184C and 80 wt % of Darocur 1173;2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure2959); methylbenzoylformate (Darocur MBF); alpha,alpha-dimethoxy-alpha-phenylacetophenone (Irgacure 651);2-benzyl-2-(dimethylamino)-1-[4-(4-morpholinyl)phenyl]-1-butanone(Irgacure 369); a mixed initiator (Irgacure 1300) of 30 wt % of Irgacure369 and 70 wt % of Irgacure 651;diphenyl(2,4,6-trimethylbenzoyl)-phosphine oxide (Darocur TPO); a mixedinitiator (Darocur 4265) of 50 wt % of Darocur TPO and 50 wt % ofDarocur 1173; a phosphine oxide; phenyl bis(2,4,6-trimethyl benzoyl)(Irgacure 819); a mixed initiator (Irgacure 2005) of 5 wt % of Irgacure819 and 95 wt % of Darocur 1173; a mixed initiator (Irgacure 2010) of 10wt % of Irgacure 819 and 90 wt % of Darocur 1173; a mixed initiator(Irgacure 2020) of 20 wt % of Irgacure 819 and 80 wt % of Darcocur 1173;bis(etha5-2,4-cyclopentadiene-1-yl)bis[2,6-difluoro-3-(1H-pyrrole-1-yl)phenyl]titanium(Irgacure 784); a mixed initiator containing benzophenone (HSP 188); andderivatives thereof. In a preferred embodiment, the photo-initiatorcomprises 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone.In other embodiments, the photo-initiator comprises2-hydroxy-3-(3,4-dimethyl-9-oxo-9H-thioxanthen-2-yloxy)-N,N,N-trimethy-1-propanaminiumchloride. Additionally these photo-initiators may be used incombination.

In a preferred embodiment the cross-linker is any chemical entity thatis able to promote the cross-linking of the polymer formed from themonomer. In one embodiment, the cross-linker comprises 1,4-bisacryloylpiperazine (BAP). In other embodiments, the cross-linker comprisesN,N′-(1,2-dihydroxyethylene)bis-acrylamide, N,N′-bis(acryloyl)cystamine,N,N′-methylenebisacrylamide, poly(ethylene glycol)diacrylate,trimethylolpropane ethoxylate triacrylate, (+)-N,N′-diallyltartramide,or mixtures thereof.

Humectants, Buffers and Other Components

In addition to the acrylic-based monomer, water-soluble organicphoto-initiator and water-soluble acrylic-based cross-linker, the enzymeimmobilization matrix can optionally further comprise other stabilizingcomponents that include, e.g., a pH buffer, a disulfide bond reducingagent, a divalent ion chelating agent, a protease inhibitor, an albumin,a salt, a sugar, a biocide, a humectant and a plasticizer. In apreferred embodiment the enzyme immobilization matrix comprises TRISbuffer, bisacrylamide, dithiothreitol, ethylene diamine tetraacetate,sucrose, aprotinin, bovine serum albumin, sodium chloride, potassiumchloride, sodium azide and glycerol. See FIG. 14.

In some embodiments, the compositions of the present invention comprisea humectant. When a humectant is added, it is preferably selected fromglycerol, propylene glycol, glyceryl triacetate, sorbitol, xylitol,maltitol, polydextrose, quillaia, lactic acid, lithium chloride and1,2-propanediol. In one embodiment, the concentration of humectant inthe composition is on the order of 2-20%, e.g., about 2-15%, about 2-10%or about 2-8% (v/v). In some cases, it has been found that at too highof a concentration, the humectant can reduce product shelf-life.Humectants, e.g., glycerol, are added to prevent the matrix from dryingduring the microdispensing process and prior to the curing step. If themicrodispensed drop dries too soon, the components of the formulationcan precipitate out of solution and this can adversely affectscross-linking and curing. As a result of the small size of the dropsdispensed onto a substrate, microdispensed in a low humiditymanufacturing environment, is easily prone to rapid drying. Accordingly,it is important to control the ambient temperature and humidity.Preferable ranges for the processes described here are 4 to 25° C. and 5to 30% relative humidity.

The formulations of the present invention preferably comprisebiochemical buffer components useful for maintaining and optimizing theenzymatic activity. Exemplary buffers includetris(hydroxymethyl)aminomethane (TRIS), sodium barbital,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), piperazinediethanesulfonic acid (PIPES), 2-(N-morpholino)ethanesulfonic acid(MES), 3-(N-morpholino)propanesulfonic acid (MOPS), Tricine, BIS-TRIS,phosphate, phosphate-saline, saline sodium citrate (SSC), saline sodiumphosphate ethylene diamine tetraacetic acid (SSPE),N-tris(hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), trisacetate ethylene diamine tetraacetic acid (TAE), tris borate ethylenediamine tetraacetic acid (TBE), and mixtures thereof. In a preferredembodiment, the buffer is TRIS.

In some embodiments, the compositions of the present inventionoptionally comprise a reducing agent. Exemplary reducing agents includedithiothreitol (DTT), 2-mercaptoethanol, tris(2-carboxyethyl)phosphineHCl, dithioerythritol, glutathione and mixtures thereof. In a preferredembodiment, the reducing agent is DTT. It may be advantageous to add areducing agent to the compositions of the presenting invention toprevent enzymes comprised in the compositions from forming inactivemultimers.

In some embodiments, the compositions of the present inventionoptionally comprise a cation binder, preferably, a divalent cationbinder. Exemplary divalent cation binders include ethylene diaminetetraacetic acid (EDTA), sodium citrate, ethylene glycol tetraaceticacid, diethylene triamine pentaacetic acid, ethylenediamine, andmixtures thereof. Such cation binders are added as metal chelators toprevent metal ion inactivation, as well as to prevent the activation ofproteases.

In some embodiments, the compositions of the present invention comprisea protease inhibitor. Exemplary protease inhibitors include aprotinin,chicken egg white cystatin, antipain, cystamine dihydrochloride,chymostatin, 3,4-dichloroisocoumarin, E-64, ebselen, Gly-Gly-Tyr-Argsynthetic peptide, leupeptin, alpha2-macroglobulin,N-alpha-tosyl-L-lysine chloromethyl ketone hydrochloride, N-alpha paratosyl-L-phenylalanine chloromethyl ketone hydrochloride, pepstatin A,pesinostreptin, epsilon-amino-n-caproic acid,4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, antithrombinIII, bdellin, complement C1 esterase inhibitor, 3,4-dichloroisocoumarin,diisopropyl fluorophosphage, elastatinal, gabexate mesylate, leupeptin,alpha2-macroglobulin, N-acetyl-glu-ser-met-asp-al,N-acetyl-ile-gly-thr-asp-al, diisopropyl fluorophosphates,Na-T-Boc-deacetylleupeptin, acetyl-pepstatin, histatin 5,Cbz-Leu-Leu-Phe-al, Cbz-Leu-Leu-Leu-B(OH)₂, lactacystin,clasto-lactacystin beta-lactone, diisopropyl fluorophosphates,phenylmethylsulfonyl fluoride, pepstatin A,D-His-Pro-Phe-His-Leu-psi-(CH₂NH)-Leu-Val-Tyr,diethylenetriaminepentaacetic acid, 1,10-phenanthroline monohydrase,phosphoramidon, diisopropyl fluorophosphates, N-acetyl-eglin C, gabexatemesylate, hirudin, Nalpha-(2-naphthalenesulfonylglycyl)-4-amidino-DL-pheylalaninepiperidide,D-Val-Leu-Lys-chloromethyl ketone, para-anlinobenzamidinedihydrochloride, ecotin, trypsin inhibitor, trypsin-chyrnotrypsininhibitor and Glu-Gly-Arg-chloromethyl ketone. In a preferredembodiment, aprotinin is added as a protease inhibitor for any proteasethat may contact the membrane at the time of blood sample analysis andalso may be present in the matrix formulation which would affect productshelf-life.

In some embodiments, the compositions of the present invention comprisea biocide. Exemplary biocides include sodium azide,2-methyl-4-isothiazolin-3-one, 5-chloro-2-methyl-4-isothiazolin-3-one,thimerosal, hypochlorite, and mixtures thereof. In a preferredembodiment, sodium azide is added as a biocide for prophylacticprotection of the formulation from microorganisms either before or afterspot curing.

In some embodiments, the compositions of the present invention comprisea plasticizer. Exemplary plasticizers include glycerol, propyleneglycol, polyethylene glycol, and mixtures thereof. In a preferredembodiment, the plasticizer is glycerol. It should be appreciated thatsome plasticizers, e.g., glycerol, can act both as plasticizers and ashumectants, thus making the compositions of the present invention moreflexible. This prevents the cured acrylic resin from cracking anddelaminating during temperature changes and other conditions that mightplace the material under stress, known as environmental stress cracking(ESC).

In some embodiments, the compositions of the present invention comprisea salt. Exemplary salts include sodium chloride, potassium chloride,sodium phosphate, potassium phosphate, and mixtures thereof. Theaddition of a low concentration of salts, preferably sodium chloride andpotassium chloride, was found to reduce the rate of delamination, i.e.,loss of membrane adhesion when subsequently contacting a calibrant fluidor blood sample. While not being bound by any theory, this improvementis believed to be due to the reduction in the difference in osmoticconcentrations between the sample and the membrane with the addition ofendogenous salt. As the ion selective electrode (ISE) is sensitive tosodium and potassium ions, the concentration was optimized to reducethis background impact on the ISE. In some embodiments, the saltconcentration is from about 0.1 mM to about 140 mM, e.g., from about 30mM to about 140 mM, from about 0.1 mM to about 1 mM, from about 20 mM toabout 50 mM, or from about 20 mM to about 40 mM. In a preferredembodiment, the compositions of the present invention comprise 0.9 mMKCl and 35 mM NaCl is used.

In some embodiments, the compositions of the present invention comprisean anhydrobiotic protectant. Exemplary anhydrobiotic protectants includesucrose, trehalose, mannitol and mixtures thereof. In a preferredembodiment, the anhydrobiotic protectant is sucrose. Sucrose ispreferably added as an anhydrobiotic protectant, to enhance membranestability so that the test cartridge in which the sensor is packagedexhibits an extended shelf-life, e.g., 6-12 months or longer. Bovineserum albumin (BSA) can also be added as it was observed to increasecartridge shelf-life and ensure good membrane adhesion.

Preparation of Compositions

In some embodiments, the compositions of the present invention aremixed, aliquoted and then stored frozen, until an aliquot is thawed andused for microdispensing. In a preferred embodiment, the monomer (e.g.,acrylamide) and the cross-linker (e.g., BAP) are mixed together insolution. To the monomer/cross-linker solution is added thephotoinitiator (e.g., Irgacure 2959). In some embodiments, it isdesirable to add the photo-initiator last, as it is the most reactiveand this precaution reduces its exposure to light. In a preferredembodiment samples were frozen at −60° C. and found to be stable for atleast 4 months. Sample freezing can range from −20 to −120° C. wherecolder temperatures are preferable. At these cryogenic temperatures, theformulation can remain stable for several years.

While the compositions of the present invention are preferably used toimmobilize enzymes, those skilled in the art will recognize that theycan also be used to immobilize other biologically active materials,instead of, or as well as enzymes, e.g., antibodies, antibody fragments,RNA, single stranded DNA and double stranded DNA. See, e.g., Rehman etal., 1999, “Immobilization of acrylamide-modified oligonucleotides byco-polymerization,” Nucleic Acids Research, 27: 649-655 (1999), which isincorporated herein by reference.

When formulating the enzyme immobilization compositions of the presentinvention, it is necessary to consider both solubility and buffering ofthe composition. Enzymes generally require an aqueous buffered solutionnear pH 7, but there are exceptions, e.g., alkaline phosphatase. Forexample, the optimum pH for urease is reported 8.0 (Wall & Laidler, TheMolecular Kinetics of the Urea-Urease System: IV The Reaction in anInert Buffer, Archives of Biochemistry and Biophysics 43: 307-311(1953)). It has been found, however, that in order to obtain an optimalenzyme activity in the compositions of the present invention, it isadvantageous to use a pH less than pH 8.0. In some embodiments the pH ofthe compositions of the present invention is from about 6.5 to about7.4. The pH of the compositions may be maintained in that range by usingwell known buffers. An exemplary buffer includes, but is not limited to100 mM TRIS, at pH 7.6. TRIS buffers ranging from 10 to 200 mM can alsobe used in the pH range from about pH 6.5 to about 8.0. Other buffersuseful in the present invention include sodium phosphate, potassiumphosphate, TRIS (trishydroxymethylaminomethane), e.g., TRIS-H₂SO₄,HEPES, TRIS-HCl buffer and barbitone.

Most photo-initiators also have limited solubility in aqueous basedsolvents. Additionally, acrylic resin cross-linkers are also onlyslightly soluble in aqueous solutions. For example, the photo-initiator2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone, whendissolved in an acrylic resin solution comprising a monomer and across-linker, was found to be slightly more soluble and could bedissolved into the aqueous solution. Higher concentrations ofphoto-initiator are preferred. It is important, however, that thephoto-initiator does not precipitate out of solution. Accordingly,identifying an appropriate concentration range is important. In someembodiments, the concentration range of photo-initiator is from about0.5 to about 10%, e.g., from about 0.5 to about 5%, or from about 0.5 toabout 4.0%.

A microdispensed layer of the preferred matrix comprising urease,2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone,bisacryloyl piperazine and acrylamide monomer is shown in FIGS. 2( c)and 3(b). The matrix is dispensed in a controlled volume onto anammonium ion-selective membrane (see FIG. 2( b)) sufficient to cover themembrane, and then exposed to sufficient UV radiation to form animmobilized urease layer adhered to the membrane. The nominal volume ofthe microdispensed matrix is preferably about 50 nL, but a wide range ofvolumes can be used. For sensors with the dimensions shown in FIG. 2,the range is preferably 10-200 nL.

FIG. 3 shows scanning electron micrographs (SEMs) that illustratetypical prints created using different protocols. With spot-curing (seeFIG. 3( b)), each microdispensed drop is exposed to UV at apredetermined time interval after the microdispensing event. Control ofthe time domain for each individual printed membrane was as follows forFIG. 3( b): t₀ dispense membrane, t₁ apply UV, t₂ stop UV, where t₀ tot₁ is 0.5 second and t₁ to t₂ is 0.6 second. For the immobilized enzymelayer shown in FIG. 3( b), the UV radiation wavelength was 310 nm, andthe UV intensity was 2 W/cm². Preferably, the dispense membrane matrixstep t₀ to t₁ ranges from about 0.05 to about 2.0 seconds, e.g., fromabout 0.1 to about 1.0 seconds. The UV radiation step preferably rangesfrom about 0.05 to 120 seconds, e.g., from about 0.1 to about 60seconds. The UV radiation wavelength can vary, for example, from about260 to about 360 nm, and preferably is specific to the photoinitiator.Preferably, with2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanonephotoinitiator, the UV wavelength is 310 nm. The UV radiation intensitycan vary from about 0.005 to about 50 W/cm², e.g., from about 0.01 toabout 10 W/cm². The UV radiation intensity and time are relatedcharacteristics of the process, wherein a reduction in one typicallynecessitates an increase in the other parameter. Further, shorterwavelengths of UV radiation can have a negative impact on sensitivebiological materials. Accordingly, wavelengths above about 300 nm arepreferred.

By way of comparison, FIG. 3( c) depicts a curing step using a flood UVsystem. The UV flood cure system requires that all the drops of matrixare microdispensed onto a substrate, e.g., a wafer, before the floodcuring step is executed. This means that the earlier drops dry (orset-up) for longer than the later drops. This delay can lead totime-dependent variations in the cured structure. Given the smalldimensions of the printed drops, they can dry quickly with thecomponents becoming insoluble, and thus are less amenable to being UVcured. For comparison, FIG. 3( a) shows an enzyme layer formed by theconventional ELVACE process. “ELVACE” is a vinyl acetate ethylenecopolymer composed of hydrophilic and hydrophobic domains. The ELVACEprocess does not involve a UV curing step. It is noted that the variablesurface using the ELVACE structure may undesirably contribute toperformance variability, as can the structure shown in FIG. 3( c).

It has been found that the UV spot cure process provides the mostconsistent domed structures, as shown in FIG. 3( b), and surprisinglyand unexpectedly yields superior sensor performance characteristics.FIG. 4 shows typical sensor output data for a spot-cured acrylamide BUNsensor, in going from calibrant fluid to blood, and FIG. 5 shows typicalsensor correlation data for the spot-cured acrylamide BUN sensor inblood. Note that these structures are also more robust mechanicallyhaving superior adhesion compared to the other structures.

In FIG. 4, the chronopotentiometric graph shows the potential differencewhen the calibrant solution is measured at time point 200, and then atest sample or in this case two different blood samples with a low andhigh urea concentration, are added to the sensor at time point 201.After a short time for the sensor output to stabilize, the potentialdifference is measured at time point 202. The difference between thepotential at time points 202 and 201 can be used to determine the ureaconcentration in the sample. This is based on the Nernst equation wherethe slope and intercept are empirically determined. The change involtage at time points 202 and 201 can be semi-log plotted against thelogarithm of analyte concentration giving a graph with a linear responseto voltage based on the analyte concentration.

In FIG. 5 the new UV cured enzyme immobilization layer (EIL) is comparedto the prior art enzyme formulated in ELVACE (also termed a film-forminglatex). Experiments used two different biosensor chips, one heated(BCL4-5) and the other un-heated (BCL3-5). Two different blood donorsamples, 169M (male) and 658F (female) were used. Some samples weretested without spiking with additional urea, and others were amended byadding urea, identified as low spike and high spike. Five cartridgeswere built for each test condition and the raw potential difference inmV was recorded. The standard deviation (SD) of the 5 samples wascalculated for each sample tested. These data showed comparable standarddeviation to the prior art ELVACE-based process.

Importantly, with the EIL process, it was found that the impact ofhaving consistently reproduced sensors is an increase in precision,reproducibility of print thickness, improved ease of manufacture andimproved product yield. For example, FIG. 18 demonstrates that for arange of print thicknesses, the potential difference measured was notsignificantly impacted by print thickness, attesting to the robustnature of this EIL process. CV1, CV2, CV3, CV4 and CV5 are standard testfluids containing concentrations of urea at 152.5, 57.8, 10.7, 5.9 and3.4 mg/dL BUN, respectively. In addition to urea, these solutions alsocontain other salts and buffering components. In FIG. 18, prints ofthickness of 57.3, 71.4, 97.3 μm were tested and graphed.

When formulating an enzyme immobilization composition comprising one ormore enzymes, an acrylic-based monomer, a water-soluble organicphoto-initiator and a water-soluble acrylic-based cross-linker in asubstantially homogeneous aqueous mixture, it is typically necessary toconsider both solubility and buffering. Enzymes generally require anaqueous buffered solution near pH 7, but there are exceptions, e.g.,alkaline phosphatase. Most photo-initiators also have limited solubilityin aqueous based solvents. Additionally, acrylic resin cross-linkers arealso only slightly soluble in aqueous solutions. The preferred1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one(manufactured by Ciba-Geigy, Irgacure 2959) when dissolved in theacrylic resin solution (monomer and cross-linker) was found to beslightly more soluble and could be dissolved into the aqueous solution.Higher concentrations of photo-initiator are preferred. It is important,however, that the photo-initiator does not precipitate out of solutionand identifying an appropriate concentration range is important.Concentrations ranging from about 0.5 to about 4.0% (w/v) are preferred.As this photo-initiator is sensitive to UV light at 310 nm, it was foundto be generally insensitive to indoor light, and was thus found to beuseful for a production process without the need for red room or yellowroom manufacturing conditions.

FIGS. 14( a) and (b) show a table of reagents for the spot curing matrixwith preferred actual mixture compositions. The first example is for aspot cured sensor with the only enzyme being urease (FIG. 14( a)), andthe second example is one for a urease and carbonic anhydrasecombination (FIG. 14( b)). In the preferred urease mixture, the order ofmixing is as follows:

Acrylic resins that are typically used for electrophoresis gels are athigh monomer to cross-linker concentration compared to the acrylic resinformulations preferably employed in the present application.Electrophoresis type resins (e.g., acrylamide and bis-acrylamide) aretypically at monomer and cross-linker concentrations of 0.2 and 0.007g/ml, respectively, whereas the preferred matrix for the presentinvention has an acrylic resin formulation containing monomer andcross-linker concentrations of 0.05 and 0.02 g/ml, respectively. Thishigher cross-linker to monomer ratio is believed to be advantageous inreducing the physical change in the microdispensed print during curingand drying. In various embodiments, the composition includes across-linker to monomer weight ratio greater than about 0.04:1, e.g.,greater than about 0.1:1 or greater than about 0.3:1. These ratios arealso advantageous to sensor hydration with a biological sample (e.g.,blood). To accomplish the desired monomer:cross-linker ratios andprevent precipitation out of solution, the typical gel electrophoresiscross-linker bis-acrylamide was replaced in the preferred embodimentwith 1,4-bis(acryloyl)piperazine (BAP), which exhibits higher aqueoussolubility.

Wafer Fabrication and Biosensors

Silicon wafers are preferably used as the solid substrate on whichbiosensor chips are created. Other materials e.g., plastics alumina andglass, can be substituted for silicon, however the former is aconvenient material for manufacture of planar structures at high volume,e.g., many millions of devices per year.

Onto the silicon, layers of materials are added at specific locations tocreate a set of individual chips. These processes are well known tothose skilled in the art. For the present invention, a thin layer ofsilicon dioxide is preferably formed over the silicon by pyrolysis. Thentitanium or a titanium-tungsten alloy is sputtered down and photoformedon top of the silicon dioxide layer. This is used as a layer for silverand other metals to adhere to. In this example silver and silver oxideare formed on the chip, as depicted in FIG. 2( a) using well knownprocesses. In FIG. 2( a), the contact pads 350 for connecting to theanalyzer device are connected to the various sensors and electricalconnectors. Specifically, the underlying layer of the BUN sensor 353contains silver and silver chloride. The chloride sensor 352 also has asilver/silver chloride layer. The ground electrode 351 is a photoformedsilver chloride electrode with multiple contact points with the sample.It forms the ground potential for electrical measurements of othersensors on the chip. The reference electrode structure is depicted by354 and described in detail in U.S. Pat. No. 4,933,048 and PublishedU.S. Appl. No. 20070015977, the entireties of which are incorporatedherein by reference. These layers preferably use several photo-definablemasks in their process to permit accurate deposition of materials atspecific locations.

FIG. 2( b) depicts a next step in the process wherein the ammoniumionophore layer 355 is deposited on top of the BUN silver/silverchloride deposition shown in FIG. 2( a). The ammonium ionophore layerand methods for its preparation are described in U.S. Pat. No.5,200,051, the entirety of which is incorporated herein by reference.This is followed by microdispensing the chloride (CL) sensor 352, shownin FIG. 2( c), along with the UV based BUN EIL membrane 356 of thecomposition described in FIG. 14.

FIG. 1 depicts in a topological manner the cross sectional layers of aBUN sensor according to one non-limiting embodiment of the presentinvention. The BUN sensor shown includes reference sensors described inthe process reflected by FIGS. 2( a)-(c). The silicon wafer 320 iscovered with a silicon dioxide layer 315, along with titanium ortitanium-tungsten alloy 310, followed by silver 305 and silver chloride304. A PVC ionophore composition 325 is printed above the silver/silverchloride layer, followed by the EIL enzyme layer 311 containing theurease enzyme.

In FIG. 1, the reference electrode also contains all the above describedlayers found up to the silver/silver chloride layer 304, but alsocontains an electrolyte layer 312, a gas permeable membrane 308 andoptionally is processed with a photoresist cap 309.

Automated Integrated Microdispensing and Spot Curing System

In a preferred aspect of the invention, the microfabricating process ofthe invention is an automated system, which is able to microdispenseprecise and programmable amounts of the materials in select regions of aplanar surface material used in the sensors of interest and additionallyprovide for integrated curing, preferably spot-curing, by means of UVradiation. The key concept in this aspect is controlling the time domainwith regard to the microdispensing step and the timing and duration ofthe subsequent UV exposure step. Here, control of the time domain istypically in fractions of seconds. This is consistent with high volumemanufacturing processes familiar for microfabricated devices.

In one embodiment, the dispensing head comprises a syringe needle with areservoir for the matrix, and a displacement means for controlling thedispensed volume from the syringe and onto the selected surface. Theapparatus also includes a step and repeat mechanism for moving thesurface, e.g., silicon wafer, with respect to both the dispensing headand said UV radiation source, thus enabling the formation of an array ofimmobilized enzyme layers at a set of pre-selected locations.

Preferably the controlled volume that is dispensed is in the range offrom about 1 nL to about 10 μL, e.g., from about 5 nL to about 1 μL orfrom about 50 nL to about 0.1 μL, and the dispensed volume will cover anarea in the range of about 10 square microns to about 75 squaremillimeters. Preferably, this area is substantially circular with radialdimensions in the range of from about 5 μm to about 5 mm.

In a preferred embodiment, the system provides a method of forming anorganized array of immobilized layers on a substantially planar surfaceby dispensing a sequence of controlled volumes of a photoformablematrix, e.g., the above-described enzyme immobilization compositions ofthe present invention, at a pre-selected set of locations on a surface.This is followed by applying UV radiation beam onto an areasubstantially covering each of the pre-selected locations. Importantly,this occurs in sequence, starting at a predetermined time after eachcontrolled volume has been dispensed and applying said radiation at apredetermined intensity for a predetermined duration, to form saidimmobilized array of immobilized layers. Preferably, the predeterminedtime is in the range of from about 0.1 to about 10 seconds, and thepredetermined duration is in the range of from about 0.1 to about 10seconds. Preferably, the method uses UV radiation in the wavelengthrange of from about 185 to about 400 nm and having an intensity in therange of from about 100 mW/cm² to about 10 W/cm². As is known by expertsin the field, curing can be effected by generating a specific dosage ofUV radiation at a selected site. It is well known that the parameters oftime, intensity and distance all impact the UV radiation dose and can beadjusted to generate a specific UV dose. Typically, a high intensity cangenerate more heat and can have other effects on the process. Thepreferred method also uses a planar surface that is a silicon wafer andthe pre-selected set of locations is an array of sensors on said wafer,typically based on unit cells in an X-Y array. In terms of thesequencing of the UV exposure after the printing step, the methodpreferably operates in a manner where the UV radiation beam is appliedto the Nth minus X pre-selected location while dispensing occurs at theNth pre-selected location. Typically, X is equal to an integer from 1 to10. In the preferred embodiment for the urease membrane (311 and 356),the parameters UV exposure parameters include a 310 nm wavelength, 0.56seconds of exposure to 4.2 W/cm² of radiation.

FIG. 6 illustrates a microdispensing system according to one embodimentof the present invention. As shown, the microdispensing system comprisesa vacuum chuck 106 and a syringe 102 and 105, each of which are attachedto separate means for altering one or more of the vertical, horizontal,lateral, or rotational displacement of these elements. For the sake ofeconomy, it is sufficient to have means for changing the verticaldisplacement of the syringe so long as one can change the position ofthe vacuum chuck multi-directionally. The movements of both elements maybe controlled via a personal computer. In one aspect, the position ofthe vacuum chuck may be reproducible within ±51 microns or better ineither or both the x and/or y directions and the flatness of the chuckis within 1 micron.

The matrix formulations of the preferred embodiments of the presentinvention can be loaded into a microsyringe assembly 102 for the purposeof establishing layers in a controllable manner. The microsyringeassembly is preferably equipped with 25 to 30 gauge needles 105 havingan internal diameter of 150 μm and an external diameter of 300 μm.Typically, the microsyringe needle 105, which includes an elongatedmember and a needle tip, is made of a metallic material, such as, forexample, stainless steel. Additional layers may be coated onto theneedle to change its surface properties. Furthermore, other materialssuch as synthetic polymers may also be employed in manufacturing themain body of the needle, itself. Depending on the pretreatment of theelectrode surface and the volume amount of fluid applied, membranelayers of a thickness ranging from about 1 to about 200 μm can beobtained consistently.

The UV cure microdispensing subsystem (FIG. 6) optionally comprises avalve 101 connected to tubing which connects to the needle holder andbarrel 102, as well as the needle 105. The microdispensed drops can beoptically monitored using a microscope 100. The microdispensed drops arecured using the UV light from the focusing lens assembly 104 usingradiation from optional light-guide fiber 103.

Non-limiting FIG. 7 shows the radiation focused in focusing lensassembly 104 using radiation from the light-guide fiber 103, whichradiation is generated by a UV bulb 151 in the UV light box 150. Asshown, the amount of light radiation and the time of exposure iscontrolled using a shutter/aperture 152 in the light box 150. The latteris optionally performed with an algorithm in a computer.

One non-limiting embodiment of the microdispense system is furtherillustrated in FIG. 8( a) where the pneumatic valve 101 generatespressure for the system along tubing 132 into the needle holder andbarrel 102. As shown, needle holding and barrel 102 is held by syringehousing 131, which holds syringe 130. Syringe 130 allows the delivery ofmicrodispensed drops through needle 105.

An optional embodiment of the UV focusing lens assembly 104 is furtherillustrated in FIG. 8( b) wherein the UV light is provided to theconcentrator housing 401 via light-guide fiber 103 through light-guide405. The concentrator housing 401 is attached to light-guide fiber 103using a mounting bracket 400. The light is focused in this example withtwo lenses 403 held in place with a lense spacer 404 in the lensebracket 402. One lense might be sufficient, but additional optionallenses are optional, and understood by those skilled in the art.

The optionally computer controlled process for the microdispensing andUV curing may be run by an algorithm depicted in FIG. 9 wherein theneedle is moved up and away from the X-Y tray which in turn is placedinto the first print position. The needle is moved down into closeproximity to the printing location, followed by print pressure beinggenerated for a brief time period. The needle is then raised after theprint pressure has finished dispensing a droplet. Concurrently with themicrodispensing process, the trailing UV cure process is started oncethe X-Y tray has positioned itself to a new print location and finishesprior to it moving again. This process is repeated for each printlocation until all positions are printed and cured.

The drop sizes that can be dispensed reproducibly extend over a widerange. For volume sizes between about 5 to about 500 nanoliters (nL),the drops can be applied preferably with a precision of about 5%. Asolenoid having a 0.1% precision rating is sufficient for this purpose.The height of the tip of the syringe needle above the sensor preferablyis between about 0.1 to about 1 mm, depending on the volume to bedispensed. Generally, the smaller the volume of the drop, the lower theelevation of the needle from the sensor. The precise alignment of thesyringe needle with the preselected area of the sensor can be achievedoptically by means of a camera and a reticle. Such an operation can beperformed manually by an operator or automatically by means of a visualrecognition system. The latter is preferred.

It is useful to consider the dynamics involved when a single drop offluid is formed and expelled from a needle. As more fluid is expelledfrom the needle tip, the drop will grow in size until the gravitationalforce acting on the mass of the drop exceeds the opposing forcesmaintaining contact with the needle tip. These opposing forces includethe adhesive forces between the needle tip and the fluid or liquid, andsurface tension of the liquid itself. It is well established that at lowliquid flow rates where discrete drop formation is complete, the dropvolume is fixed. However, the volume may be changed by varying any ofthe fluid related parameters discussed above, or by changing thediameter of the needle tip thus changing the available surface area forfluid adhesion. For example, a hydrophobic polytetrafluoroethylene(PTFE) coating applied to the needle tip reduces the natural drop sizeof an aqueous based matrix material by reducing the adhesive forcesbetween the drop and the needle tip. In circumstances where a controlledvolume must be microdispensed onto a surface, it is possible to have themicrosyringe tip positioned above the planar surface at a height whichdoes not allow the drop to form completely (and then fall to the surfaceunder the influence of gravity), but the partially formed drop actuallycontacts the surface and the new adhesive forces between the liquid andthe surface begin to spread the drop. If the needle tip is now retractedin the Z-direction a sufficient distance away from the surface, then thecohesive forces of the liquid is overcome and a volume of liquid lessthan the fixed drop size will remain in contact with the surface. Thistechnique can be used to dispense reproducibly any volume of liquid fromabout one-one thousandth of the fixed drop size and greater.

The surface tension between a pure liquid and its vapor phase can bechanged by adding reagents. For example, a fatty acid added to waterreduces the surface tension, whereas added salts can increase surfacetension. The microdispensable fluid compositions of the presentinvention preferably are prepared to have a controlled optimized surfacetension. Suitable additives may be used when necessary. Thehydrophobicity or hydrophilicity of the fluid is controlled in the samemanner. Where a cured membrane is required as the end product, thesolids content and volatile solvents content preferably are carefullyadjusted. Moreover, the ratio of these components is also used tocontrol the viscosity.

The preferred microdispensable compositions for the ammonium ion sensorcomprises PVC polymer, plasticizers, ionophores and solvents withviscosities generally higher than those used for planar casting (e.g.,spin-coating) of membranes. These higher viscosity compositions cure ordry without deformation of the membrane layer. Related problems, e.g.,that of ensuring the homogeneity of the matrix at high viscosity andthus preventing phase separation of materials after time (i.e.,considerations related to shelf-life) are also alleviated by thesecompositions. Other additives are also used to prevent long-termdegradation of the membranes.

In addition to the factors described above relating to controlledvolumetric dispensing of fluids having an optimized surface tensionassociated with a prescribed composition, tailoring the surface freeenergy of the substrate, or surface onto which the fluid is dispensed,provides control over the final dimensions, especially the thickness, ofthe resulting layer. The resulting process is highly versatile, allowingthe deposition of arrays of layers of varied composition and utility.For establishing thick membranes, (e.g., 40-60 μm thick), the surface ispreferably tailored so that the contact angle which the microdispensedfluid makes with the surface is large. For example, before an aqueousbased enzyme matrix is microdispensed, the surface may be first plasmatreated to give a controlled contact angle. For the preferred ureasematrix, a carbon tetrafluoride plasma step yields a contact angle in therange 50°-70°.

An improved aspect of the microdispensing system, described here, is theintegration of an automatic spot curing component. An EXFO Omnicure UVsystem is preferred for integration due to its ability to continuallymonitor and adjust the light aperture to assure that the radiationintensity remained consistent throughout the process. This amelioratesthe issue of a typical UV bulb intensity decreasing over its lifetime(˜2000 working hours) by using 50% intensity as the set point. As thereis a relationship between cure time and bulb intensity, a reasonablyhigh setting is required to reduce the product processing time.

Another aspect of the UV cure process is the desire to focus the beam onthe specific sensor to avoid UV exposure to other sensors. Focusing thebeam needs to be appropriate to avoid being too limiting. This isbecause the visualization system used to align each sensor that is beingprocessed needs enough flexibility to assure a robust process in theevent that they are not accurately aligned. Intensity is related to thedistance of the UV beam to the cure site, therefore, by being closer theintensity is increased and the product processing time is decreased.

While the invention is described primarily in terms of a silicon waferwith microfabricated ion-selective electrodes, other types of sensorscan be fabricated to incorporate a surface onto which the disclosedcomposition can be dispensed or coated. These include optical sensors,fiber optic sensors, surface acoustic wave sensors, evanescent sensors,surface plasmon resonance sensors and optical wave guide sensors. Italso includes various base sensors, e.g., electrodes, ion-selectiveelectrodes, potentiometric electrodes, amperometric electrodes,conductimetric electrodes, enzyme electrodes, biosensors, opticalsensors, fiber optic sensors, surface acoustic wave sensors, evanescentsensors, surface plasmon resonance sensors and optical wave guidesensors. Substantially planar surfaces for sensor fabrication caninclude silicon wafers, alumina wafers, liquid crystal substrates, glasssubstrates and plastic substrates and flexible plastic substrates. Inpreferred embodiments, membrane-forming compositions are exposed tosufficient UV radiation to cause significant cross-linking, thus formingan adhered non-swelling immobilized enzyme layer on the surface.

The integrated microdispense and UV cure device is preferablyautomatically programmed in order to optimize the manufacturing processtime and to effect UV curing. The microdispensing and UV curing stepspreferably are run in tandem. In a preferred aspect, the UV curing steptakes approximately 0.5 seconds, whereas the microdispense step takesabout 0.3 to 0.4 seconds. Therefore, the microdispense step is typicallyrate limited by the indexing time of approximately 0.1 seconds betweenprint sites. The microdispense and the UV cure subsystems preferablyoperate at two different, but adjacent physical locations during thesame time period, wherein the microdispense step occurs before and aheadof the UV cure operation. FIG. 9 provides a preferred algorithm foroperation.

The dispensing apparatus with the integrated UV radiation sourcepreferably has a registration and alignment means capable of focusing abeam of radiation onto an area substantially covering the location atwhich a drop of matrix has been dispensed. A computer means is able toswitch the UV radiation on and off, and this occurs at a predeterminedtime and for a predetermined duration (and also at a predeterminedintensity), after the matrix has been dispensed. The registration andalignment means permits a beam to be focused on a selected area of saidsurface and illuminate an area in the range of about 10 square micronsto about 75 square millimeters.

Each wafer preferably is manufactured with a plurality of chips(typically about one thousand on a 5 inch wafer), each containing one ormore sensors and in this case each containing the BUN sensor. Thesesensors are desirably arranged in a uniform X-Y arrangement on thewafer. For processing of the wafers in the preferred embodiment, thechips are preferably generated by first placing an adhesive tape on theback of the wafer followed by cutting the wafer into individual chipsusing, for example, a diamond dicing saw. This process causes a slightdisplacement and uneven arrangement compared to the original location ofthe chip on the wafer. To compensate for this, a microscope 100, asdepicted in FIG. 6, is used along with visual recognition software torealign the chip for the microdispense process. For the initialalignment and registration of the UV cure system, a UV sensitive paperis used to determine the proper alignment. Alternatively, a visiblelight source can be inserted in place of the UV light source 151 abovethe light guide 103 in order to align and focus the UV cure site. Yetanother alternative approach would be use a UV monitoring device(radiometer) which has a focusing point and the intensity and activityis used to align and focus the UV cure site. Note that alternatively thedicing step can be performed after dispensing and spot-curing, howevercare is required to ensure that the water coolant used for the dicingblade does not result in dicing dust damaging the cured membranes. Wherethe substrate is plastic rather than a silicon wafer, dicing is by asimpler cutting process where dust damage is not an issue. Here dicingafter dispensing and spot-curing is preferred.

Cartridge Construction for the Use of Improved Sensors

The diced silicon chips described above are then preferably used assubcomponents for disposable plastic cartridges. Each cartridgetypically contains several features allowing it to process a patientsample with an analyzer device and determine the presence or amount ofan analyte, e.g., urea, in the sample.

Referring to the figures, the cartridge for accepting chips of thepresent invention comprises a cover (two views), FIGS. 10, 11, a base,FIG. 13, and a thin-film adhesive gasket, FIG. 12, disposed between thebase and the cover and securing them together. Specifically, thebackside of the cover shown in FIG. 10 mates with the exposed face ofthe gasket of FIG. 12, and the backside of the gasket mates with theexposed face of the base of FIG. 13. Referring now to FIG. 10, the cover1 is made of a rigid material, preferably plastic, and capable ofrepetitive deformation at flexible hinge regions 5, 9, 10 withoutcracking. The cover comprises a lid 2, attached to the main body of thecover by a flexible hinge 9. In operation, after introduction of asample into the sample holding chamber 34, the lid can be secured overthe entrance to the sample entry port 4, preventing sample leakage bymeans of deformable seal 11, and the lid is held in place by hook 3. Thecover further comprises two paddles 6, 7, that are moveable relative tothe body of the cover, and which are attached to it by flexible hingeregions 5, 10. In operation, when operated upon by a pump means, paddle6 exerts a force upon an air bladder comprised of cavity 43, which iscovered by thin-film gasket 21, to displace fluids within conduits ofthe cartridge. When operated by a second pump means, paddle 7 exerts aforce upon the gasket 21, which can deform. The cartridge is adapted forinsertion into a reading apparatus, and therefore has a plurality ofmechanical and electrical connections for this purpose. It should alsobe apparent that manual operation of the cartridge is possible. Thus,after insertion of the cartridge into a reading apparatus, the readingapparatus transmits pressure onto a fluid-containing foil pack filledwith approximately 130 μL of calibrant fluid located in cavity 42,rupturing the package upon spike 38, and expelling fluid into conduit39, which is connected via a short transecting conduit in the base tothe sensor conduit, 16. When the calibrant fluid contacts the sensors,they wet-up and establish a signal associated with the amount ofcalibrating ion or molecule in the fluid.

Referring to FIG. 12, thin-film gasket 21 comprises various holes andslits to facilitate transfer of fluid between conduits within the baseand the cover, and to allow the gasket to deform under pressure wherenecessary. Holes 30 and 33 permit one or more urea sensors and one ormore reference electrode that are housed within either cutaway 35 or 37,to contact fluid within conduit 16.

Referring to FIG. 13, conduit 34 is the sample holding chamber thatconnects the sample entry port 4 to first conduit 16 in the assembledcartridge. Cutaways 35 and 37 are locations in the housing for acceptingthe chips of the present invention. Optionally they also house aconductimetric sensor for determining the position of air-liquidboundaries. Recess 42 houses a fluid-containing package, e.g., arupturable pouch, in the assembled cartridge that is pierced by spike 38because of pressure exerted upon paddle 7 upon insertion into a readingapparatus. Fluid from the pierced package flows into the second conduitat 39 and then into conduit 16. An air bladder is comprised of recess 43which is sealed on its upper surface by gasket 21. The air bladder isone embodiment of a pump means, and is actuated by pressure applied topaddle 6 which displaces air in conduit 40 and thereby displaces thesample from sample chamber 34 into conduit 16.

Improved Method of Forming and Curing Membranes Arrays

In one embodiment, the method of manufacture of a BUN sensor requirestwo separate printing events. The first step involves an NH₄ ⁺ion-selective electrode (ISE) print step followed by a urease layerprint step. As described above, the printing process preferably uses apneumatic pump and valve with a fine gauge needle on top of an X-Y tableto accurately microdispense a drop onto a specific location on a wafer.Several controllable factors contribute to overall sensor performance.These include: (i) accurate registration of the printed drop over thedesired location, (ii) the appropriate viscosity and surface tension ofthe drop, (iii) the associated hydrophobicity of the surface, (iv) theheight and width (or volume) of the drop, (v) the height of the dropafter drying on the surface, and (vi) good adhesion. Furthermore, theformation of crystals (partitioning) or cracking of the dried dropletcan adversely affect sensor performance.

The prior art enzyme membrane (see, e.g., U.S. Pat. No. 5,200,051) iscomposed of a film-forming latex, preferably ELVACE (Forbo AdhesivesSynthetic Polymers, Morris, Ill.). However, this heterogeneous materialcan be susceptible to drying and blocking the microdispensing needle tipwhich can have an adverse affect on manufacturing. The surface tensionof the ELVACE can also create irregular shaped structures which mayimpact the performance of the sensor. See FIG. 3( a). Additionally,ELVACE, which is a vinyl acetate ethylene (VAE) copolymer composed ofhydrophilic and hydrophobic domains, can degrade over time, most likelygenerating acetate which makes the material more acidic. This timedependent process reduces the usable lifetime of the material. As aresult, it is desirable to replace this heterogeneous matrix with asubstantially more stable composition, preferably a homogeneous aqueousmatrix which can be photoformed and provides a stable immobilizationenvironment for enzymes, e.g., urease.

The present invention solves several lifetime issues including: thelifetime of the raw materials, the lifetime of the aqueous matrix priorto print, and the lifetime of the printed and cured matrix in thecompleted sensor. It also survives contacting a blood sample in thefinal product without dissolving away. This provides evidence of goodadhesion characteristics that are highly desirable for reliable sensorperformance.

Most importantly for a reliable manufacturing method, the presentinvention provides a microdispensable matrix that remains in solution atstorage temperatures in the range of about 4° C. to about 35° C. withoutprecipitation of the sub-components. It can also be stored frozen andmelted for use without deleterious effect. Advantageously, the matrixcomposition also has adequate UV transmission, printed at thicknesses ina range extending to about at least 200 μm, to have a high degree ofpolymer conversion throughout the matrix.

The present invention is advantageous as the matrix exists in a state oflow viscosity during the printing process, and then is controllablyconverted by UV radiation into a gelled or fixed state. This controlledapproach using a UV cure process requires the incorporation of a UVphoto-initiator in the formulation. This also requires a curing systemthat can generate controlled UV radiation directed to the part beingcured. This specification of this UV curing system needs to provide for(i) simple automation, (ii) operation on a short cycle time compatiblewith the printing system, (iii) avoiding racking diced wafers during thecuring process, (iv) avoiding the need for an oven or heat curing step,and (v) avoiding the need for continuous matrix mixing. In addition, itis desirable to avoid unnecessary material wastage. As previouslymentioned, from a manufacturing perspective it is also useful if a largebatch of material is made, pre-aliquoted and stored in frozen form.

The present invention permits a robust manufacturing process. Theaspects of a robust manufacturing process permit some range of precisionat each step in the process while generating a consistent productresult. For one step in the process, the print thickness can varyslightly. From data (see FIG. 18), the mean potentiometric signal wasadvantageously found to be independent of the thickness of the print.Further, the standard deviation of these values across a lot of wafersis fairly independent of print thickness. This allows a wide range ofenzyme layer thicknesses without impacting product performance.

Various UV systems may be used including a light wand, UV flood curewith or without an oven step, and a light-guide system. A UV laser canalso be used for this process to deliver a focused beam of radiation tothe printed sensor site. For the process of curing printed membranes ona silicon wafer or similar substrate, e.g., glass and plastic, a lightwand is required to be positioned over every print location. This canincrease the cure step time by n x t, where n is the number ofchips/print locations and t is the time in seconds of each UV cure step.In one embodiment, a UV cure step is provided by a system accuratelymoving a light wand to each successive print location. The light wandmovement is integrated with the print event at each print location.

For a system where the UV source moves independent of the print head,the matrix is first printed at a specific location by the print needle.The print needle moves away from the surface, preferably in the Zdirection. The UV source then moves into location, followed by a UV cureexposure at the print location.

In a system where the UV source trails by a fixed off-set from the printsite, the print needle prints in the fashion of a typewriter style printprocess (e.g., from left to right, followed by a return to the left sideand an index to the next line of print sites, which are again processedfrom left to right). A UV source is located one or more print sitesaway, n, from the print needle location. The print needle prints thefirst print site, and then another print site until it reaches n printsites. Trailing along behind the print needle is the UV source. Once theUV source is located at the first print site on the left, n sites awayfrom the print needle, the UV source exposes and cures the print site.As the print needle moves to the next print site, the UV source moves tothe next site and while the print needle dispenses the matrix, the UVsource exposes the print site. This print and trailing UV cure processcontinues until the print needle reaches the right side of the planarsurface. In order to finish the UV cure process, the needle continues tomove to the right with the associated movement of the UV source whichcontinues to cure the remaining n sites. The print needle may beprogrammed to stop printing for the remaining print sites. The printneedle and UV source are indexed to the next row to be printed and thesetup begins at the left side of the planar surface. It should beunderstood by those skilled in the art that the print needle and UVsource could be fixed in their position, and the table holding theplanar surface moved to effect the movement of the print needle and UVsource to each individual print site. Those skilled in the art willrecognize that other engineered registration means can be used toaccomplish the objective of ensuring consistent control of the timedomain, such that each dispensed layer is cured at a fixed time and in afixed manner after it is printed.

In the preferred embodiment a light-guide is used. The light-guide iseffectively a conduit for light where the light is to be focused onto asmall region of a wafer and where it is not delivered in a straight linefrom the UV source. The UV radiation is directed along the light-guideby a light fiber connection. This has the advantage of a light wandsystem where the lamp and filters are contained in the power box and the“flashlight” uses a very small footprint at the location of the curesite. Additionally, the UV radiation contains less attendant heat frominfrared radiation and therefore keeps the cured part cool.

Where the particular photoinitiator present in the matrix requiresspecific radiation wavelengths, this is achieved by selection ofspecific UV bulbs appropriate for that specific photoinitiator. Moststandard UV bulbs generate multiple wavelengths, some desired and somenot necessarily desired. In applications with biological materials, itis preferred to have a specific radiation wavelength with no additionalextraneous radiation at other wavelengths. Note that it is well known inthe scientific literature that UVC radiation is less damagingbiologically than UVA or UVB. Therefore, avoiding or limiting thesewavelengths is preferable for biological samples. In the preferredmatrix formulation which uses Irgacure 2959, the preferred radiationwavelength is about 310 nm. Common UV bulbs use mercury (type “H”) andmetal halide (“D”), which can both be used to cure as they generate UV Aand UV B radiation required for the Irgacure 2959 photoinitiatorcontaining matrix. However the “H” bulb has less extraneous radiationwavelengths and is preferred.

It is beneficial for the UV system to have an integrated optical filterallowing the passage of specific wavelengths of non-ionizing radiation,while preventing the transmittance of undesired wavelengths. In thepreferred embodiment, radiation near 310 nm is required for thephotoinitiator. As a means to limit the exposure of the sensor todeleterious wavelengths of radiation, a narrowband filter such as theGilway and International Light Technologies, Inc. (Peabody, Mass.)Narrowband Filter NS313 which efficiently only permits wavelengths fromabout 300 to 340 nm is useful for preferred embodiments. Other filtersused singly or in combination will be apparent to those skilled in theart to affect appropriate radiation wavelengths specific for certainother photoinitiators.

In a preferred embodiment, the light-guide system (FIGS. 6, 7 and 8 b)has the advantages of spot curing with the capability to direct light toa specific location. In addition it can easily be filtered and does notgenerate the heat found in flood exposure based systems filteringInfrared (IR) radiation. It also does not require a separate shuttersystem, as this feature is already integrated into the device. Thisapproach is also desirable for integration with the microdispensingsystem, as the power supply can be positioned outside of themicrodispense housing with only the light-guide and concentrator housingwith its associated lenses inserted inside the microdispensing unitminimizing footprint.

It is desirable that the UV-curable enzyme matrix has the followingcharacteristics: (i) compatibility with enzymes and particularly for thepreferred embodiment the enzyme urease enzyme, (ii) exhibit theappropriate flow and viscosity for good printing characteristics, (iii)compatibility with a reliable chemical formulation which can support ahigh-yield manufacturing process, e.g., incorporate an antimicrobialagent for improving matrix shelf-life and reagents for stabilizing anenzyme, (iv) be a water based technology to support biological reagents,(v) achieve reliable adhesion to a surface after UV curing step, (vi)provide for rapid wet-up when used in conjunction with a sensor, (vii)exhibit the appropriate enzyme substrate and water permeability whenformed as a layer to support and sustain the enzyme reaction (viii)exhibit good electrical characteristics when used with anelectrochemical sensor, and (ix) show an extended post-processinglifetime of greater than about 6 months at room temperature or underrefrigeration, e.g., be compatible with genuine commercial productrequirements. The compositions described in FIG. 14 have these desiredcharacteristics.

The UV curing systems described herein can be used with each UV curableformulation to characterize many operating parameters including theprecision and accuracy of the dimensions of cured membranes and adhesionof the membrane to the surface. The sensor can then be tested incartridges to determine sensor performance with a given matrix andcuring combination. This can include intra-wafer and inter-wafervariations, where each wafer may contain as many as a thousand sensors.

In FIG. 16 the effect of radiation on the sensor performance based onthe matrix of FIG. 14( a). Batches of sensors were prepared using aflood lamp process and processes with 0.5 s, 1 s, 2 s and 3 s spot-cureUV radiation with radiation doses of 190, 380, 760 and 1140 mJ,respectively. These data show that the processes are robust with variousconditions of radiation giving acceptable test results.

FIG. 15 demonstrates that compositions containing alternative monomersand crosslinkers generated similar sensor signals with test solutions.The monomers tested included acrylamide, methacrylamide, poly(ethyleneglycol)acrylate (PEGA), and N-[3-(Dimethylamino)propyl]-methacrylamide(DMAPMA). Additionally, the crosslinkers tested included1,4-bis(acryloyl)piperazine, polyethylene glycol diacrylatepoly(ethylene glycol)diacrylate (PEGDA),N,N′-(1,2-dihydroxyethylene)bis-acrylamide (DHEBA) andtrimethylolpropane ethoxylate triacrylate (TMPETA).

FIG. 17 shows an experiment where the absence of the humectant glycerolunder accelerated lifetime storage conditions (e.g., 30° C. or 40° C.)had an affect on performance. By adding glycerol to the present matrix(see compositions in FIG. 14), a process with good performance andshelf-life is achieved.

Improved Blood Urea Nitrogen Sensor Manufacture

In one aspect, the intent of the present invention is to improve on themanufacture of BUN (blood urea nitrogen) sensors. The preferredembodiment of the new BUN sensor is manufactured using a combination ofthin-film microfabrication processes and microdispensing techniques. Itcomprises a thin film silver-silver chloride indicator electrodeoperating in combination with a thin-film silver-silver chloridereference electrode of the type described in U.S. Pat. No. 4,933,048,incorporated by reference herein. A more preferable reference electrodeis described in Published U.S. Appl. No. 20070015977, incorporated byreference herein.

In the initial step, a substrate wafer of silicon is overlaid with aninsulating layer of silicon dioxide, by thermal oxidation. Metal layersof a titanium and tungsten alloy (TiW) and then silver are subsequentlydeposited onto the silicon dioxide base wafer and then patterned usingphotolithographic techniques. An electrically insulating layer such aspolyimide polymer or additional silicon dioxide is then photo-patternedto isolate adjacent sensor circuitry. The silver-silver chlorideindicator electrode (diameter ˜200 microns) is prepared from thepatterned silver using standard techniques, e.g., electrochemical,chlorine gas plasma and oxidation of Ag⁰ by an inorganic oxidant such asCr₂O₇ ²⁻ or Fe³⁺ in the presence of chloride ion.

The remaining layers of the BUN electrode include two thick-filmstructures: (i) a semi-permeable membrane film, comprising an organicpolymer layer (e.g., poly(vinyl chloride)—PVC), and an ammonium ionionophore; and (ii) the outermost biolayer, comprising in thisparticular sensor, a spot photo-cured acrylamide urease layer thatoptionally includes carbonic anhydrase. These layers are deposited by amicrodispensing technique as described in U.S. Pat. No. 5,554,339,incorporated by reference herein. In the present invention, however, themicrodispensing assembly described in the '339 Patent has beensubstantially improved to include an integrated ultraviolet spot-curingcomponent system enabling automated printing and curing in a controlledtime domain, as described above.

The thick-film ammonium ion-sensitive structure comprises a poly(vinylchloride) (PVC) binder, tris(2-ethylhexyl)phosphate as a plasticizer,and nonactin as the ionophore. The indicator electrode can be madeselective for different ions by using the same (or similar) binder andplasticizer composition but with different ionophores. For example,valinomycin, monensin and (methyl)monensin, and tridodecylammoniumchloride have been used to make potassium, sodium, or chloride-ionselective electrodes, respectively. Other ionophores may include, butare not limited to crown ethers, trialkylamines, or phosphate esters,and the like. Alternatively, other polymeric binder materials may beused besides PVC. These polymers may include, for example, siliconrubber, polytetrafluoroethylene plastics, or derivatives of PVCcontaining ionizable functional groups (e.g., carboxylates). Otherplasticizers suitable for use in the present invention may include, butare not limited to tris(2-ethylhexyl)phosphate, nitrocymene,2-nitrophenyloctyl ether, dibutyl sebacate, diethyl adipate, phthalates,propylene carbonate, 5-phenylpentanol, or mixtures thereof. Still otherbinders and ionophore combinations may occur to those skilled in theart, which are within the scope of the present invention. The resultingsemi-permeable ion-selective film may have a thickness in the range ofabout 2 microns to about 200 microns, preferably about 10 to about 30microns. In the preferred embodiment, the ammonium ion-selectivemembrane solvent system is selected to provide the appropriate surfacetension and stability. The solids content (wt %) of plasticizer, PVCpolymer, and ionophore are preferably 60-80%, 15-40% and 0.5-3%,respectively.

Various methods can be used to define a layer on a planar substrate. Ifa thick layer (about 5 to about 200 microns) is required,microdispensing of a viscous matrix, e.g., the photo-curable ureasematrix described above, is generally preferred. Other methods fordefining a layer on a planar substrate include, without limitation,spin-coating, dip-coating, spray coating, screen printing, ink-jetprinting, laser printing, painting and contact printing are alternativemethods and may be better suited to a different applications. Forexample, the preferred urease photoformable matrix may be screen printedonto a wafer in a single pass at a specific time (t=0). The screenoptionally has an opening of 300 μm diameter, with each openingregistered for alignment with an array of ammonium ion-selectivemembranes on the wafer. After the printing step a flood UV exposure ofthe wafer is performed at t=1. Similarly to the spot-curing methoddescribed above, this method also gives control of the time domain fromprinting to the UV step for each individual matrix. In this embodimentt=1 is preferably automatically set at 2-20 seconds after t=0. Automatedequipment for moving wafers from a printing station to an exposurestation is well known in the microfabrication art. The preferred ureasematrix can also be spin-coated and photo-patterned with a mask, in themanner widely used in the microfabrication art.

Referring now to the topological illustration in FIG. 1, the substratewafer, 320, is silicon, with an overlaid insulating layer of silicondioxide, 315. In addition there is a polyimide layer 301 with twocircumferential print wells (302, 303) which are used to confine theprinted layers. The first metal layer, 310, is TiW and serves thefunctions of a conductor and an adhesion layer to the wafer. Succeedinglayers 305 and 304, are the silver and silver chloride layers. On theleft side of FIG. 1, the remaining layers of the indicator electrodeinclude (i) a semi-permeable membrane film, 325, comprising an organicpolymer layer (e.g., polyvinyl chloride (PVC)) and an ammonium ionionophore; and (ii) the outermost biolayer, 311, comprising in thisparticular embodiment, an acrylamide photo-cured urease layer of thepreferred composition described above.

The reference electrode portion of the unit cell may be comprised ofoverlaid structures as shown in FIG. 1. In this particular embodiment,the metal and chloridized layers of the reference electrode are coveredby an electrolyte layer, which may comprise any material which is ableto hold a high concentration of salt but which is, preferably,photoformable. In this respect, a polyvinyl alcohol (PVA) formulation isthe preferred material and may first be photo-patterned and forms awater-permeable matrix that can subsequently be saturated with a salt,such as potassium chloride. A separate gas permeable membrane, may alsobe present which serves to diminish the loss of electrolyte or salt tothe bulk analytical sample but allows the rapid wet-up (i.e., passage ofwater or other small gaseous molecules) of the reference electrode priorto commencing the sample analysis.

The patterning process can be either of those described in U.S. Pat. No.4,933,048 and Published U.S. Appl. No. 20070015977 both incorporatedherein by reference. Alternatively, a reference electrode structure canbe used in which the distance between the liquid junction and thesurface of the silver/silver chloride is sufficiently large, such thatthe concentration of electrolyte in the immediate vicinity of theAg/AgCl structure is substantially constant for a period of timesufficient to perform a measurement of the potential difference betweenthe indicator electrode and the reference electrode.

Referring now to FIG. 2, indicator electrode and the adjacent referenceelectrode are each connected by an overpassivated signal line to acontact pad. The overpassivation (polyimide layer) includes print wells302 and 303 formed as concentric circles. The unit cell is confinedwithin a rectangular area, which is repeated in an array several hundredtimes on a single silicon wafer. In particular embodiments of theinstant invention, other indicator electrodes may be present in the unitcell for the simultaneous measurement of other species (e.g., Na⁺, K⁺,Cl⁻) in addition to ammonium ion.

To manufacture the BUN base sensor, a silicon wafer with a topical layerof silicon dioxide, which had previously been cleaned, scrupulously witha mixture of concentrated sulfuric acid and hydrogen peroxide is placedinto a plasma deposition system and layers of TiW (0.1 μm) and silver(0.5 μm) are sputtered consecutively onto the wafer surface. Thesilver-titanium bilayer is then processed to localize it to a region,which in the final device acts as the ammonium ion sensor. This processis achieved by a standard lithographic technique in which the wafer isspin-coated with positive resist (Shipley AZ 1370 SF). After UV exposureof the photoresist through a mask and development (Shipley AZ 351), theexposed silver is removed by an aqueous solution of ferric nitrate (0.9mM) as the etchant. The underlying titanium layer is then processed bymeans of the same photolithographic steps, but using an aqueous mixtureof nitric acid (3.9M) and hydrofluoric acid (0.78 M) as the etchant.N-methylpyrrolidone solvent is then used to remove the remainingphotoresist to expose the required silver structures (diameter about 150μm).

To passivate the signal lines a photo-definable polyimide (DuPont 2703)is spin-coated onto the wafer. Once the wafer is UV exposed anddeveloped with a solvent the polymer is baked in an oven at 350° C. for30 minutes under an inert atmosphere and left to cool to 150° C. beforeremoval. While the mask used for patterning defines the perimeter of thelayer, it also defines print wells 302 and 303. These are subsequentlyused to control the dimensions of the two respective microdispensedmembranes.

The silver is preferably then chloridized by dipping the entire waferinto an aqueous solution of potassium dichromate (12 mM) andhydrochloric acid (60 mM). The wafer is then washed and partially diced.Over these patterned silver chloride electrodes is placed an ammoniumion sensitive membrane. The membrane material is made by dissolving lowmolecular weight PVC (Sigma) and high molecular weight carboxylated PVC(Type Geon, Goodrich) (1:1 w/w) in a solvent system of cyclohexanone,propiophenone, and N-methylpyrrolidone (1:1:1 v/v/v) to a total solidscontent of 10 g/dL of solution. Dissolution is accomplished by heatingthe mixture at 70° C. for 30 minutes. To this mixture the plasticizertris(2-ethylhexyl)phosphate (Fluka) is added, to provide a total solidscontent of 35 g/dL. The resulting mixture is then allowed to cool to 45°C. and nonactin (Fluka) is added in the amount equivalent to 2 percentof the total solids in the mixture. At room temperature, 10-100 nL ofthis final material is microdispensed onto each of the silver chlorideindicator electrodes on the wafer, overlapping on all sides by at leastabout 30 μm. Print well 302 is preferably used to define the perimeter.Curing is accomplished by placing the wafer on a 60° C. hot-plate for 30minutes. This process yields a stable, rugged structure having athickness of about 15 μm.

In the final step the preferred urease matrix, described above, ismicrodispensed onto individual membranes and UV spot cured using theapparatus. Print well 303 is preferably used to define the perimeter. Asmentioned above, in the preferred formulation the components are mixedtogether and frozen in a cryofreezer prior to use. This allowsconsistent production of product day-to-day and a long storage lifetimeprior to microdispensing. This formulation can also be quality control(QC) tested prior to a microdispense event as the mixture can be assayedfor enzymatic activity using a standard reference method using aspectrophotometer. This reduces the cost and waste in making product inthe manufacturing process.

Regarding the preferred standard assay method, the urease enzymeactivity from an aliquot of the thawed frozen formulation is assessedusing a modification of the method of Kaltwasser & Schlegel,“NADH-dependent coupled enzyme assay for urease and otherammonia-producing systems,”Analytical Biochemistry 16: 132-138 (1966).The method measures the spectrophotometric change of NADH to NAD at 340nm in a glutamate dehydrogenase assay coupled to urease.

Cartridge Analyses Using the Improved BUN Sensor

In the preferred embodiment, the finished chips containing the sensorsare then assembled into test cartridges and used to make BUNmeasurements in blood. One embodiment of a cartridge of the presentinvention is shown in FIGS. 10-13.

The cartridge is preferably adapted for insertion into a readingapparatus, and therefore has a plurality of mechanical and electricalconnections for this purpose. It should also be apparent that manualoperation of the cartridge is possible. Thus, after insertion of thecartridge into a reading apparatus, the reading apparatus transmitspressure onto a fluid-containing foil pack filled with approximately 130μL of calibrant fluid located in cavity 42, rupturing the package uponspike 38, and expelling fluid into conduit 39, which is connected via ashort transecting conduit in the base to the sensor conduit, 16. Whenthe calibrant fluid contacts the sensors, they wet-up and establish anelectrical signal associated with the amount of calibrating ion ormolecule in the fluid.

Referring to FIG. 12, thin-film gasket 21 comprises various holes andslits to facilitate transfer of fluid between conduits within the baseand the cover, and to allow the gasket to deform under pressure wherenecessary. Holes 30 and 33 permit one or more urea sensors and one ormore reference electrode that are housed within either cutaway 35 or 37,to contact fluid within conduit 16. Referring to FIG. 13, conduit 34 isthe sample holding chamber that connects the sample entry port 4 tofirst conduit 34 in the assembled cartridge. Cutaways 35 and 37optionally houses a conductimetric sensor for determining the positionof air-liquid boundaries. Recess 42 houses a fluid-containing package,e.g., a rupturable pouch, in the assembled cartridge that is pierced byspike 38 because of pressure exerted upon paddle 7 upon insertion into areading apparatus. Fluid from the pierced package flows into the secondconduit at 39 and then into conduit 16. An air bladder is comprised ofrecess 43 which is sealed on its upper surface by gasket 21. The airbladder is one embodiment of a pump means, and is actuated by pressureapplied to paddle 6 which displaces air in conduit 40 and therebydisplaces the sample from sample chamber 34 into conduit 16.

In the preferred embodiment, the BUN sensor is packaged into a cartridgeof the type disclosed in U.S. Pat. No. 5,096,669, which also contains acalibrant solution. It is contained in a calibrant package (cal-pack),which is ruptured during the blood sample analysis. The typical sequenceof events includes the cal-pack being ruptured and then the calibrationsolution passing over the sensor and wetting up the sensor. Typically,the cal-pack of prior art cartridges contained the following ions,sodium, potassium, calcium, chloride, bicarbonate and also HEPES buffer,glucose, lactate, urea, creatine and creatinine. As a diagnostic testfor research purposes, urea was replaced with ammonia. This permitsstudies of the ammonium ionophore performance independent of the ureasecontaining enzyme immobilization layer. The preferred constitution ofthe cal-pack for the new BUN sensor preferably include sodium,potassium, calcium, chloride, urea, HEPES buffer, glucose, lactate,creatine and creatinine.

A potentiometric chemical sensor for urea can be viewed as a system,which is constructed from functionally dissimilar components. In oneembodiment of the blood urea nitrogen (BUN) sensor, the outermost layer,the one in contact with the analyte solution, permits the transport ofurea while also serving to immobilize the active enzyme molecules. Theseenzymes catalyze the hydrolysis of urea to ammonia as described above.At neutral pH values, the ammonia thus produced exists predominantly asammonium ions. By interposing a separate layered structure, whichcontains an ionophore with high sensitivity and selectivity for ammoniumions between the enzyme containing layer and a silver-silver chlorideindicator electrode, the ammonium ion concentration at the electrodeinterface can be measured. In this type of measurement, the potentialdifference between the indicator electrode and a reference electrode isrecorded. This is done with a potentiometric circuit in an instrument(or analyzer) which makes connection with the two electrodes, as is wellknown in the electrochemical measurement art. The analytical value ofthe measurement is derived from the fact that the magnitude of thepotential difference is related by the Nicolsky equation to theconcentration of the analyte, in this case, urea:E=E _(o) +RT/nF log[A+Σ(a,b)k(a,b)B]where E is the measured electromotive force (signal), R is the gas lawconstant, T is the absolute temperature, n is the absolute value of thecharge on analyte species a (e.g., n=1 for the ammonium ion), F is theFaraday constant, A is the activity of the analyte species a, B is theactivity of an interfering chemical species b, k(a,b) is theinterference coefficient associated with the effect of the presence ofchemical species b on the electrochemical potentiometric determinationof the activity of the analyte species a, and E_(o) is a constantindependent of T, A, or B. See Amman, D., Ion-Selective Microelectrodes,Springer, Berlin (1986) p. 68, and references cited therein, which isincorporated by reference herein.

Data presented in FIGS. 4, 5 and 15-18 were recorded and/or analyzed inusing these principles. These data are presented using a commercial BUNtesting system for comparison. The performance of the new sensor issuperior to the established technology.

EXAMPLES Example 1

A solution of aprotinin is prepared by dissolving 0.01 g of aprotinin in50 mL of deionized water, generating a 0.02% concentration stocksolution. An enzyme buffer solution is prepared by the addition of 4.32g of glycerol, 130 g of deionized water, 130 g of 1M TRIS at pH 7.6, 2.6g of 0.5M EDTA at pH 8.0, 13 g of the above 0.02% aprotinin stocksolution, 0.2 g of 1,4-dithioerythritol, 2.7 g of sodium chloride, 0.097g of potassium chloride, 0.05 g of sodium azide, 92.8 g of sucrose and29 g of BSA. The mixture is vortexed until fully dissolved. A solutioncontaining the acrylic resin components is then prepared, including 72.2g of acrylamide, 150 g of deionized water, 29.4 g of1,4-bis(acryloyl)piperazine and 2.5 g of activated carbon. This acrylicresin solution is mixed until in solution and filtered using a 0.2 umfilter into a clean container. To this solution 17.8 g of1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one isadded, mixed and the solution is kept from excessive exposure to lightin a container wrapped in aluminum foil. Finally 40 g of urease is addedto about 333 g of the above filtered solution and mixed. About 44 g ofdeionized water is added, and finally 184 g of the acrylic resinsolution (prepared as above) is added. The material is kept covered inaluminum foil and mixed until in solution. For long term storage, thissolution is aliquoted in 1 ml portions and frozen at −60° C.

Example 2

In another embodiment of the urease enzyme immobilization layer includedthe enzyme carbonic anhydrase. The order of mixing is as follows: Asolution of aprotinin is prepared by dissolving 0.01 g of aprotinin in50 mL of deionized water, generating a 0.02% concentration stocksolution. An enzyme buffer solution is prepared by the addition of 4.32g of glycerol, 130 g of deionized water, 130 g of 1M Tris at pH 7.6, 2.6g of 0.5M EDTA at pH 8.0, 13 g of the above 0.02% aprotinin stocksolution, 0.2 g of 1,4-dithioerythritol, 2.7 g of sodium chloride, 0.097g of potassium chloride, 0.05 g of sodium azide, 92.8 g of sucrose, 29 gof BSA and vortexed until the contents are fully dissolved. A solutioncontaining the acrylic resin components is then prepared, including 72.2g of acrylamide, 150 g of deionized water, 29.4 g of1,4-bis(acryloyl)piperazine, and 2.5 g of activated carbon. This acrylicresin solution is mixed until in solution and filtered using a 0.2 umfilter into a clean container. To this solution 17.8 g of1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one isadded, mixed and the solution is kept from excessive exposure to lightusing aluminum foil. Finally 40 g of urease and 14 mg of carbonicanhydrase is added to about 333 g of the above filtered enzyme solutionand mixed. About 44 g of deionized water is added, and finally 184 g ofthe acrylic resin solution prepared above is added. The material is keptcovered in aluminum foil and mixed until in solution. This solution isaliquoted in 1 ml portions and frozen at −60° C.

Any feature described or claimed with respect to any disclosedimplementation may be combined in any combination with any one or moreother feature(s) described or claimed with respect to any otherdisclosed implementation or implementations, to the extent that thefeatures are not necessarily technically incompatible, and all suchcombinations are within the scope of the present invention. Furthermore,the claims appended below set forth some non-limiting combinations offeatures within the scope of the invention, but also contemplated asbeing within the scope of the invention are all possible combinations ofthe subject matter of any two or more of the claims, in any possiblecombination, provided that the combination is not necessarilytechnically incompatible.

1. A method of forming an immobilized layer on a sensor on asubstantially planar surface comprising the steps of: (a) dispensing acontrolled volume of a photoformable matrix at a pre-selected locationon said surface, wherein the photoformable matrix comprises abiologically active material selected from the group consisting ofprotein, enzyme, antibody, antibody fragment, RNA, single stranded DNAand double stranded DNA, an acrylic-based monomer, a water solubleorganic photo-initiator and a water soluble acrylic-based cross-linkerin an aqueous mixture; and (b) applying a UV radiation beam onto an areasubstantially covering said pre-selected location, starting at apredetermined time after said volume has been dispensed and for apredetermined duration at a predetermined intensity, to form saidimmobilized layer.
 2. The method claim 1, wherein the immobilized layeris an enzyme layer.
 3. A method of forming an array of immobilizedlayers on an array of sensors on a substantially planar surfacecomprising the steps of: (a) dispensing a sequence of controlled volumesof a photoformable matrix at a pre-selected set of locations on saidsurface, and (b) applying a UV radiation beam onto an area substantiallycovering each said pre-selected location, in sequence, starting at apredetermined time after each controlled volume has been dispensed, andapplying said radiation at a predetermined intensity for a predeterminedduration, to form said immobilized array of layer wherein the UVradiation bean is applied to the Nth minus X pre-selected location whiledispensing occurs at the Nth pre-selected location, where X is equal toan integer from 1 to
 10. 4. The method of claim 3, wherein thepredetermined time is in the range of about 0.1 to about 10 seconds. 5.The method of claim 3, wherein the predetermined duration is in therange of about 0.1 to about 10 seconds.
 6. The method of claim 3,wherein said UV radiation is in the wavelength range of about 185 to 400nm.
 7. The method of claim 3, wherein said UV radiation intensity is inthe range of about 100 to 400 mW/cm².
 8. The method of claim 3, whereinsaid planar surface is a silicon wafer and the pre-selected set oflocations is an array of sensors on said wafer.